Andrographis paniculata and Silybum marianum are well known medicinal
plants. However, to prove their efficacy for clinical utilization more scientific
data are needed. Therefore, in the present study, an attempt was made to investigate
the anticancer potential of hydroalcoholic extracts of A. paniculata
and S. marianum and their combination (1:1). The sulphorhodamine B (SRB)
assay was used to assess growth inhibition of human tumor. Five human cancer
cell lines i.e., human breast adenocarcinoma (MCF-7), human cervix (SiHa), Colon
(HT-29), Ovary cancer cell line (ovcar-5) and Liver (HepG2) were used for the
above study. The results obtained suggest that S. marianum hydroalcoholic
extract showed the best cytotoxic activity against all given cell lines with
percentage inhibition of 21.34, 32.30, 46.56, 59.58, 36.20 for MCF 7, SiHa,
HT-29, Ovcar-5 and HepG2, respectively. While, A. paniculata hydroalcoholic
extract was found most effective against Ovcar-5 with 51.12% inhibition. The
combination of both the plants (1:1) showed an intermediate result for all the
cell line but, it was found to be most effective against HepG2 with 42.76% inhibition.
The results obtained in the study indicate that A. paniculata and S.
marianum possess significant anticancer activity and have the therapeutic
potential to prevent the cancerous diseases.

Cancer is the uncontrolled growth and division of cells and it leads to a mass of cells known as tumor. In the body normal cells complete their life cycle by following an orderly path of growth, division and death. The process of programmed cell death is called apoptosis. When apoptosis breaks down, it leads to cancer. Chemotherapy is a major clinical treatment used for the control of advanced stages of malignancies and metastasis. But many cancer patients seek alternative and complementary methods of treatment because of the serious side effects of chemotherapy and radiation therapy. Several herbs and plants with diversified pharmacological properties have been shown to have potential to prevent human cancers as they possess antitumor substances which have the potential to cure cancer without causing toxicity.

Extract preparation: Andrographis paniculata and Silybum marianum dried and powdered aerial part were extracted separately with 70% ethanol using soxhlet apparatus. The filtrate was evaporated to dryness using a rotary evaporator (yield was 16.83% w/w and 15.04% w/w) for A. paniculata and S. marianum, respectively. The extracts were then stored below ambient temperature for further studies.

Phytochemical test: Phytochemical analysis was performed to detect various compounds such as tannins, flavonoids, alkaloids, steroids etc.

Experiment: The human tumor cytotoxicity was determined by the protocols
established by NCI (Monks et al.,1991). The Sulphorhodamine
B (SRB) assay was used in this study to assess growth inhibition. The colorimetric
assay estimated cell number indirectly by staining total cellular protein with
the dye SRB. Single-cell suspensions were prepared by the treatment of cells
with 0.5-1 mL of 0.1% trypsin-EDTA. The viable cells were counted using a Coulter
counter and diluted with RPMI medium and final densities of 100x104
cells mL-1 were obtained. Cell suspensions (100 μL well-1)
was seeded in 96-well microtiter plate containing 1 mL of media and incubated
for cell attachment. After 24 h, the cells were treated with the extracts. Extracts
were initially dissolved in 100% DMSO (1 mg mL-1 for extracts) and
further diluted in RPMI medium to produce a concentration of 100 μg mL-1.
For combinational study (1:1) ratio, concentrations of the extracts were prepared
by dissolving 50 μg of A. paniculata and 50 μg of S. marianum
in 1 mL of DMSO. The control wells were filled with 100 μL of medium and
plates were incubated for 48 h.

After 48 h, adherent cell cultures were fixed by adding 50 μL of cold 50% (w/v) trichloroacetic acid (TCA) and incubating for 60 min at 4°C. The supernatant was then discarded and the plates were washed 5 times with deionised water and then dried. Each microtiter well was added with 100 μL of SRB solution (0.4% w/v in 1% acetic acid) and the culture was incubated for 30 min at room temperature for complete staining reaction. Unbound SRB was removed by washing five times with 1% acetic acid and then the plates were air-dried. Bounded stain was solubilised with Tris buffer (10 mM) and the optical densities were read on an automated spectrophotometric plate reader (Molecular Devices, USA) at a wavelength of 492 nm, Optical density (OD) of SRB in each well is directly proportional to the cell number.

Statistical analysis: The experiment was performed in triplicates and the data are given as mean SRB absorbance for the calculation of inhibition±SEM. Percentage inhibition was calculated by comparing the OD of control well with that of test sample:

RESULTS

Phytochemical study showed the presence of flavonoids, tannins, carbohydrate,
cardiac glycosides, terpenes and steroids in both the plant extracts. Apart
from this, alkaloids were present only in the extract of Andrographis paniculata
while, saponins were present only in Silybum marianum extract (Table
1). The preliminary examination was done by observing the confluency of
the test as well as control samples which showed cell growth inhibition in the
test samples (Fig. 1, 2).

On the basis of measurement of OD, the anticancer study suggest that S.
marianum extract showed the best cytotoxic activity against all given cell
lines (percentage inhibition was 21.34, 32.30, 46.56, 59.58, 36.20 for MCF 7,
SiHa, HT, Ovcar and HepG2, respectively), while significant one was against
HT and Ovcar. Andrographis paniculata extract was found most effective
against Ovcar cell line (51.12%). Again, the combination (1:1) of both the plants
showed an intermediate result for most of the cell line but the combination
was most effective against the liver cell line (HepG2) which showed better activity
(42.76%), as compared with the activity of both the plant extracts which gave
individually (28.22 and 36.20% for A. paniculata and S. marianum,
respectively) (Table 2).

DISCUSSION

Medicinal plants maintain the health and vitality of individuals and cure various diseases, including cancer without causing toxicity. These medicinal plants possess good immunomodulatory and antioxidant properties, leading to anticancer activities. The antioxidant phytochemicals protect the cells from oxidative damage. Thus, consuming a diet rich in antioxidant plant material can provide health-protective effects. These natural products are supposed to minimize DNA damage by reacting with free radicals and in this way they can prevent cancer.

Phytochemicals, such as flavonols and flavonoids were investigated to determine
chemoprevention activity against cancer (Conese and Blasi,
1995). Phenols, polyphenols, flavonoids and their derivatives are ubiquitous
in plants and have been found associated with the inhibition of atherosclerosis
and cancer (Cirla and Mann, 2003). Recent studies have
reported antitumor effects of the flavonoids, quercetin, genistein and baicalein
obtained from plant extracts (Shoeb et al., 2006).
Similarly, alkaloids like schischkinnin and montamine have been isolated from
the seeds of Centaurea schischkinii and Centaurea montana which
showed anticancer property (Yang and Wang, 1993). Flavonoids
isolated from Plantago species were able to strongly inhibit the proliferation
of human cancer cell lines (Galvez et al., 2003).
Similarly, A. paniculata and S. marianum showed anticancer activity
due to the presence of flavonoids, alkaloids, polyphenols which may have inhibited
the protein synthesis either by damaging the DNA or by blocking at transnational
level which accounted for the mortality of the cancer cells.

In conclusions, the result supports the use of the plant as described in folk
medicine, that the aerial plant parts can be used to treat cancer. Further studies
are required to isolate the active constituents involved in the antioxidant
and hepatoprotective activity of the plant.